Advances in heparin structural analysis by chromatography technologies

doi:10.3724/SP.J.1123.2022.07020

Abstract

Abstract:

Heparin (Hp) is the most widely used anticoagulant drug in the clinics, with an annual global output of over 10 billion dollars. Hp, a member of the glycosaminoglycans (GAGs), is prepared from porcine intestinal mucosa via extraction, separation, and purification. Hp is a linear polysaccharide with repeating disaccharide units. Low-molecular-weight heparins (LMWHs) are depolymerized from Hp via chemical or enzymatic degradation. Compared with Hp, LMWHs exhibit less bleeding side effect, milder immunogenicity, and higher bioavailability when injected subcutaneously. In general, Hps, including LMWHs, are high complex drugs with large molecular weights (MWs), inhomogeneous MW distributions, and structural heterogeneity, including different degrees and locations of sulfonation, and unique residues generated from different production processes. Thus, developing efficient analytical methods to elucidate the structures of Hps and characterize or quantitate their properties is extremely challenging. Unfortunately, this problem limits their quality control, production optimization, clinical safety monitoring, and new applications. Research has constantly sought to elucidate the complicated structures of Hp drugs. Among the structural analysis and quality control methods of Hp currently available, chromatographic methods are the most widely studied and used. However, no literature thoroughly summarizes the specific applications of chromatographic methods in the structural analysis, manufacturing process, and quality control of Hp drugs. This paper systematically organizes and describes recent research progresses of the chromatographic methods used to analyze Hp drugs, including the identification and composition of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The applications, innovations, and limitations of these chromatographic methods are also summarized in this review. The insights obtained in this study will help production and quality control personnel, as well as drug researchers, obtain a deeper understanding of the complex structures of Hp drugs. This paper also provides a comprehensive reference for the structural analysis and quality control of Hps, proposes ideas for the development of new quality control methods, and lays a strong foundation for the in-depth structural elucidation of Hp drugs.

Key words: chromatography, monosaccharide composition analysis, disaccharide composition analysis, oligosaccharide analysis, polysaccharide analysis, heparin (Hp), low molecular weight heparin (LMWHs), review

CLC Number:

O658

OUYANG Yilan, YI Lin, QIU Luyun, ZHANG Zhenqing. Advances in heparin structural analysis by chromatography technologies[J]. Chinese Journal of Chromatography, 2023, 41(2): 107-121.

Figures/Tables 10

References

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No.	Derivatization reagent	Method	Parameters	Analytes	Ref.	Published year
1	PMP	RPLC-UV	LOD:0.04-1.6 μmol/L;LOQ:0.15-1.6 μmol/L;mass recovery:92%-100%;RSD:0.3%	10 sugars	[51]	2016
2	PMP	RPLC-MS(MRM)	LOD:0.056-5.6 fmol/L;LOQ:0.5-10 ng/mL;RSD:6.0%	16 sugars	[53]	2017
3	2-AB and 2-AP	RPLC-UV	LOD:1.2-11 nmol/L;LOQ:4-36 nmol/L;repeatability:2%-9%;inter-day repeatability:3%-9%	10 sugars	[52]	2021
4	d3-4-MOBHA·HCl	RPLC-MS(MRM)	LOD:1.2-11 nmol/L;LOQ:0.25-3 fmol/L;mass recovery: 85%-110%	12 sugars	[58]	2021
5	no	IC	LOD:1.0 ng;LOQ:2.5 ng	16 sugars	[45]	2012

No.	Derivatization reagent	Method	Parameters	Analytes	Ref.	Published year
1	PMP	RPLC-UV	LOD:0.04-1.6 μmol/L;LOQ:0.15-1.6 μmol/L;mass recovery:92%-100%;RSD:0.3%	10 sugars	[51]	2016
2	PMP	RPLC-MS(MRM)	LOD:0.056-5.6 fmol/L;LOQ:0.5-10 ng/mL;RSD:6.0%	16 sugars	[53]	2017
3	2-AB and 2-AP	RPLC-UV	LOD:1.2-11 nmol/L;LOQ:4-36 nmol/L;repeatability:2%-9%;inter-day repeatability:3%-9%	10 sugars	[52]	2021
4	d3-4-MOBHA·HCl	RPLC-MS(MRM)	LOD:1.2-11 nmol/L;LOQ:0.25-3 fmol/L;mass recovery: 85%-110%	12 sugars	[58]	2021
5	no	IC	LOD:1.0 ng;LOQ:2.5 ng	16 sugars	[45]	2012

No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]	IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]		HILIC- MS^Eno[60]
1	ΔU-GlcNAc	1	1	1	1	1		1		1
2	ΔU-GlcNS	1	1	1	1	1		1		1
3	ΔU-GlcNAc,6S	1	1	1	1	1		1		1
4	ΔU2S-GlcNAc	1	1	1	1	1		1		1
5	ΔU-GlcNS,6S	1	1	1	1	1		1		1
6	ΔU2S-GlcNS	1	1	1	1	1		1		1
7	ΔU2S-GlcNAc,6S	1	1	1	1	1		1		1
8	ΔU2S-GlcNS,6S	1	1	1	1	1		1		1
9	ΔGalA-GlcNS	1	0	0	0	0		1		1
10	ΔGalA-GlcNS,6S	1	0	0	0	0		1		1
12	IdoA2S-GlcNS	1	0	0	0	0		1		1
13	U2S-GlcNS or U-GlcNS,6S	0	0	1	0	0		0		0
14	U2S-GlcNAc or U-GlcNAc,6S	0	0	0	1	0		0		0
15	IdoA2S-GlcNS,6S	1	0	0	0	0		1		1
16	ΔU2S-GlcN,6S	0	0	0	0	0		1		1
17	ΔU2S-GlcN	0	0	0	0	0		1		1
18	ΔU-GlcN,6S	0	0	0	0	0		1		1
19	ΔU-GlcN	0	0	0	0	0		0		1
20	ΔGlyser	1	1	1	1	0		1		1
No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]		IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]	HILIC- MS^Eno[60]
21	ΔGlyser_ox1	1	0	0	0		0		1	1
22	ΔGlyser_ox2	1	0	0	0		0		0	0
23	Other non-endogenous derivatives ΔI_SO3	1	0	0	0		0		0	0
24	Tetrasaccharide A ΔI_S-I_2SO3	1	0	0	0		0		0	0
25	Tetrasaccharide B ΔI_2SO3-I_sid	1	0	0	0		0		0	0
26	ΔU-GlcNAc,6S-GlcA-GlcNS,3S	1	0	0	0		0		0	0
27	ΔU-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	1	1	1		1		1	1
28	ΔU-GlcNS,6S-GlcA-GlcNS,3S,6S	1	1	1	0		0		1	1
29	ΔU2S-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
30	ΔU2S-GlcNS,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
31	GlcNS-IdoA2S-GlcNS,6S or GlcNS,	0	0	1	0		0		0	0
	6S-IdoA2S-GlcNS
32	GlcNS,6S-IdoA2S-GlcNS,6S	1	0	0	0		0		1	1
33	ΔU-GlcNS,6S-HexA	0	0	0	0		0		1	1
34	ΔU2S-GlcNAc,6S-HexA	0	0	0	0		0		0	1
35	ΔU2S-GlcNS,6S-HexA	0	0	0	0		0		1	1
36	ΔU2S-GlcNS,6S-HexA2S	0	0	1	0		0		1	1
37	ΔU2CS-GlcNS,6S	0	0	0	0		0		1	1
38	GlcNS,6S-U2CS-GlcNS or GlcNS-U2CS-GlcNS,6S	0	0	0	1		0		0	0
39	ΔUA2S-GlcNS,6S-GlcA-2,3-anhydro-GlcNS	0	0	0	0		0		1	1
40	ΔUA-GlcNS-HexA2S,3S-GlcNS	0	0	0	0		0		1	1
41	3-O-S Δdp2	0	0	0	0		0		1	0
42	3-O-S Δdp4(3S,1Ac)	0	0	0	0		0		1	0
43	3-O-S Δdp4(4S,0Ac)	0	1	1	0		0		1	0
44	3-O-S Δdp4(4S,1Ac)	0	2	0	0		0		0	0
45	3-O-S Δdp4(5S,0Ac)	0	3	0	0		0		0	0
46	ΔU- Mnt6S	0	0	0	1		0		1	0
47	ΔU2S- Mnt6S	0	0	0	1		0		1	0
48	GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		0	0
49	ΔU2S-GlcNS-U-Mnt6S	0	0	0	0		0		1	0
50	ΔU2S-GlcNAc-U2S-Mnt6S	0	0	0	1		0		0	0
51	ΔU2S-GlcNS-U2S-Mnt6S	0	0	0	1		0		1	0
52	ΔU2S-GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		1	0
53	MntΔdp5(5S,1Ac)	0	0	0	1		0		0	0
54	MntΔdp6(6S,1Ac)	0	0	0	1		0		0	0
55	ΔU-1,6-anhydroGlcNS	0	0	0	0		0		0	1
56	ΔU-1,6-anhydroManNS	0	0	0	0		0		0	1
57	ΔU2S-1,6-anhydroHexNS	0	0	0	0		2		0	1
58	GlcNS,6S-U2S-1,6-anhydroHexNS	0	0	1	0		0		0	0
59	ΔU2S-GlcNS,6S-U2S-1,6-anhydroManNS	0	0	0	0		1		0	1

No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]	IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]		HILIC- MS^Eno[60]
1	ΔU-GlcNAc	1	1	1	1	1		1		1
2	ΔU-GlcNS	1	1	1	1	1		1		1
3	ΔU-GlcNAc,6S	1	1	1	1	1		1		1
4	ΔU2S-GlcNAc	1	1	1	1	1		1		1
5	ΔU-GlcNS,6S	1	1	1	1	1		1		1
6	ΔU2S-GlcNS	1	1	1	1	1		1		1
7	ΔU2S-GlcNAc,6S	1	1	1	1	1		1		1
8	ΔU2S-GlcNS,6S	1	1	1	1	1		1		1
9	ΔGalA-GlcNS	1	0	0	0	0		1		1
10	ΔGalA-GlcNS,6S	1	0	0	0	0		1		1
12	IdoA2S-GlcNS	1	0	0	0	0		1		1
13	U2S-GlcNS or U-GlcNS,6S	0	0	1	0	0		0		0
14	U2S-GlcNAc or U-GlcNAc,6S	0	0	0	1	0		0		0
15	IdoA2S-GlcNS,6S	1	0	0	0	0		1		1
16	ΔU2S-GlcN,6S	0	0	0	0	0		1		1
17	ΔU2S-GlcN	0	0	0	0	0		1		1
18	ΔU-GlcN,6S	0	0	0	0	0		1		1
19	ΔU-GlcN	0	0	0	0	0		0		1
20	ΔGlyser	1	1	1	1	0		1		1
No.	Composition	SAX- UV^Hep[63]	SAX- SEC^Hep[65]	SAX- SEC^Eno[66]	SAX- SEC^Nadro[66]		IPRP- MS^Eno[70]		HILIC- MS^Nadro[72]	HILIC- MS^Eno[60]
21	ΔGlyser_ox1	1	0	0	0		0		1	1
22	ΔGlyser_ox2	1	0	0	0		0		0	0
23	Other non-endogenous derivatives ΔI_SO3	1	0	0	0		0		0	0
24	Tetrasaccharide A ΔI_S-I_2SO3	1	0	0	0		0		0	0
25	Tetrasaccharide B ΔI_2SO3-I_sid	1	0	0	0		0		0	0
26	ΔU-GlcNAc,6S-GlcA-GlcNS,3S	1	0	0	0		0		0	0
27	ΔU-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	1	1	1		1		1	1
28	ΔU-GlcNS,6S-GlcA-GlcNS,3S,6S	1	1	1	0		0		1	1
29	ΔU2S-GlcNAc,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
30	ΔU2S-GlcNS,6S-GlcA-GlcNS,3S,6S	1	0	0	0		0		1	0
31	GlcNS-IdoA2S-GlcNS,6S or GlcNS,	0	0	1	0		0		0	0
	6S-IdoA2S-GlcNS
32	GlcNS,6S-IdoA2S-GlcNS,6S	1	0	0	0		0		1	1
33	ΔU-GlcNS,6S-HexA	0	0	0	0		0		1	1
34	ΔU2S-GlcNAc,6S-HexA	0	0	0	0		0		0	1
35	ΔU2S-GlcNS,6S-HexA	0	0	0	0		0		1	1
36	ΔU2S-GlcNS,6S-HexA2S	0	0	1	0		0		1	1
37	ΔU2CS-GlcNS,6S	0	0	0	0		0		1	1
38	GlcNS,6S-U2CS-GlcNS or GlcNS-U2CS-GlcNS,6S	0	0	0	1		0		0	0
39	ΔUA2S-GlcNS,6S-GlcA-2,3-anhydro-GlcNS	0	0	0	0		0		1	1
40	ΔUA-GlcNS-HexA2S,3S-GlcNS	0	0	0	0		0		1	1
41	3-O-S Δdp2	0	0	0	0		0		1	0
42	3-O-S Δdp4(3S,1Ac)	0	0	0	0		0		1	0
43	3-O-S Δdp4(4S,0Ac)	0	1	1	0		0		1	0
44	3-O-S Δdp4(4S,1Ac)	0	2	0	0		0		0	0
45	3-O-S Δdp4(5S,0Ac)	0	3	0	0		0		0	0
46	ΔU- Mnt6S	0	0	0	1		0		1	0
47	ΔU2S- Mnt6S	0	0	0	1		0		1	0
48	GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		0	0
49	ΔU2S-GlcNS-U-Mnt6S	0	0	0	0		0		1	0
50	ΔU2S-GlcNAc-U2S-Mnt6S	0	0	0	1		0		0	0
51	ΔU2S-GlcNS-U2S-Mnt6S	0	0	0	1		0		1	0
52	ΔU2S-GlcNS,6S-U2S-Mnt6S	0	0	0	1		0		1	0
53	MntΔdp5(5S,1Ac)	0	0	0	1		0		0	0
54	MntΔdp6(6S,1Ac)	0	0	0	1		0		0	0
55	ΔU-1,6-anhydroGlcNS	0	0	0	0		0		0	1
56	ΔU-1,6-anhydroManNS	0	0	0	0		0		0	1
57	ΔU2S-1,6-anhydroHexNS	0	0	0	0		2		0	1
58	GlcNS,6S-U2S-1,6-anhydroHexNS	0	0	1	0		0		0	0
59	ΔU2S-GlcNS,6S-U2S-1,6-anhydroManNS	0	0	0	0		1		0	1